Cellular transplantation is a recently developed biomedical technology for the study and treatment of human diseases characterized by cell dysfunction or cell death. For many such diseases current medical therapies or surgical procedures are either inadequate or nonexistent. Cellular therapy can replace or augment existing tissue to provide restorative therapy for these conditions. Exemplary cell types suitable for transplantation include: neural tissue derived cells, hepatocytes, myocytes, retinal cells, endocrine cells, melanocytes, keratinocytes, and chondrocytes. It has been shown in both animal models and in human studies that engraftment of transplanted cells can successfully reestablish tissue function.
In a specific example, cells derived from neural tissue have been used to affect the course of Parkinson""s disease, a disorder characterized by depletion of dopaminergic neurons. In neurotoxin-induced, dopamine deficient monkey and rat animal models, xenogeneic and allogeneic fetal ventral mesencephalon (VM) cell preparations isolated from pigs, rats, or humans have been reported to reverse the movement disorder (see e.g., Kopyov et al. 1992 Transplantation Proceedings 24: 547-548; Huffaker et al. 1989 Exp. Brain Res. 77:329). Human fetal VM grafts have also been reported to affect the course of Parkinson""s disease in man by reinervating the dopamine (DA) depleted host striatum (see e.g., Lindvall et al. 1990 Science, 242: 574-577; Lindvall et al. 1994 Ann. Neurol. 35: 172-180; Widner et al. 1997 Ann. Neurol. 42:95.; Kordower et al. 1995 New Engl. J. Med 332:1118).
Nevertheless, current methods of cell transplantation, particularly those which utilize freshly prepared neural tissue, have been hindered by the lack of available cell sources and limited viability of neural cells after preparation. These problems are compounded by the logistical problems involved in ensuring that surgeons, operating rooms, patients, and fresh cells are all available at the same time. Finally, the need to rapidly implant the fresh cells following preparation hinders extensive quality control prior to implantation.
In view of the above, it is desirable to store, and sometimes pool, freshly harvested cells prior to implantation. It would also be desirable to store cells which have been cultured in vitro. Such storage would allow banking, quality control, and other desired procedures and manipulations, either in connection with in vitro analysis or implantation in vivo.
Methods for cell storage prior to transplantation include preserving the tissue by freezing cells (xe2x80x9ccryopreservationxe2x80x9d) (see e.g., Chanaud et al. 1987 Neurosci. Lett. 82: 127-133; Collier et al. (1987) 436: 363-366) or by refrigerating the cells at above freezing temperatures (xe2x80x9chibernationxe2x80x9d) (see e.g., Sauer et al. 1991 Neurology and Neuroscience 2: 123-135; Gage et al. 1985 Neurosci. Lett. 60: 133-137). However, freezing of fresh neural tissue results in poor viability after thawing and low yield or recovery of cell numbers. Specifically, studies comparing survival of cryopreserved and fresh transplanted tissue have shown dramatic decreases in the survival of cryopreserved grafts as compared to fresh control tissue (see e.g., Jensen et al. 1984 J. Comp. Neurol. 227: 558-68). For example, tissue culture experiments have indicated that cryopreservation may lead to decreases in neuronal survival to between one-and two-thirds of fresh control values (see e.g., Collier et al. 1988 Progress in Brain Research, 78: 631-6). In particular, human tissue may be more susceptible to damage induced by freezing than rat tissue, which is reflected in poor graft survival and reduction in cell size of the cryopreserved neurons (Frodl et al. 1994 Brain Research, 647:286-298).
Storage of tissue in preservative media at temperatures above freezing temperatures (hibernation) can result in high rates of graft survival and function as compared to cryopreserved tissue (see e.g., Sauer et al. 1991 supra.; Kawamoto et al. 1986 Brain Res., 384: 84-93). However, cells cannot be maintained for long periods of time under such conditions. Specifically, cell viability is progressively decreased during hibernation. Within about one week, such losses render the cell population unacceptable for transplantation in vivo.
In addition, prior art methods for freezing and hibernating cells utilize complex media comprising buffers and added protein, sometimes including entirely undefined components, such as serum. However, to minimize toxicity and immunogenicity such additives are not desirable for transplantation into humans and hinder controlled studies of neural cell growth, development and function in vitro.
This invention solves the problems referred to above by providing methods for storing neural cells without significant decreases in cell viability and/or functionality. Such methods greatly enhance the availability of cells for in vitro analysis and/or transplantation in vivo. Such methods are also useful when pooling of cells is desired.
In one aspect, the invention pertains to a method for storing cells in a cryopreserved state in which fresh or cultured neural cells are suspended in a cryopreservation solution, the temperature of the cell suspension is decreased in a controlled manner to about xe2x88x92196xc2x0 C. and the cells are maintained in a frozen state.
In another aspect, the invention pertains to a method for storing cells in hibernation in which fresh, cultured, or cryopreserved neural cells are suspended in a hibernation medium which is preferably free of added protein, free of a buffer, or free of added protein and a buffer, and the cell suspension is maintained at temperatures which are above freezing and sufficiently below normal body temperature such that normal physiological cell processes are decreased or halted.
In another aspect, the invention pertains to cultures of cells which have been stored according to a cryopreservation method and/or hibernation method of the invention. Such cells are useful for in vitro growth, development, and analysis as well as for transplantation in vivo.
In another aspect, the invention pertains to methods of implantation which utilize neural cells which have been stored according to the methods disclosed herein.
The present invention pertains, inter alia, to improved methods of storing neural cells, preferably dissociated neural cells, prior to their use in transplantation and to the cells obtained using such methods. This invention provides for long-term storage of neural cells without significant decreases in cell viability and/or function. Accordingly, the present invention represents a significant advance over the previous cell storage methods. The instant invention is based, in part, on the discovery that neural cells can be stored and/or frozen in a medium, which lacks any buffer or added protein. In addition, improved methods for storing porcine cells are provided. One aspect of the invention features methods which employ a hibernation step of as long as 3-5 days prior to and/or post freezing (or instead of freezing) while still recovering neural cells suitable for transplantation. The ability to store cells without loss of viability and/or function allows for the separation in time between cell preparation and implantation into a subject. The ability to store cells also enables the pooling of cells from multiple donors and adequate time for quality control assessment of cells and other in vitro analysis.
As used herein, the following terms and phrases shall be defined as follows:
xe2x80x9cStoringxe2x80x9d includes maintaining neural cells after harvest from a donor and prior to use in transplantation in a subject. The term xe2x80x9cstoringxe2x80x9d is meant to include holding or maintaining cells either above or below freezing.
xe2x80x9cCryopreservationxe2x80x9d includes preservation of cells at temperatures below freezing.
xe2x80x9cHibernationxe2x80x9d includes preservation of cells at temperatures above freezing and sufficiently below normal physiological temperature such that one or more normal cellular physiological processes are decreased or halted. Preferred hibernation temperatures range between 0 and 4xc2x0 C., preferably about 4xc2x0 C.
xe2x80x9cNeural cellsxe2x80x9d includes any differentiated neural cells derived from the nervous system of a human or an animal, preferably the central nervous system. Preferably, neural cells for use in the instant methods have undergone final maturation, but have not sent out projections (e.g., axons). The term xe2x80x9cneural cellsxe2x80x9d includes for example, neural stem cells or neural progenitor cells which have been induced to differentiate into neural cells in vitro and neural stem cells or neural progenitor cells which have differentiated into neural cells in vivo. The term xe2x80x9cneural cellxe2x80x9d also includes, for example, neurons, astrocytes, and oligodendrocytes. The term xe2x80x9ccellsxe2x80x9d is used interchangeably herein with the term xe2x80x9cneural cellsxe2x80x9d. The term xe2x80x9ccellsxe2x80x9d as used herein encompasses both neural cells in the form of tissue (e.g., intact pieces of tissue) and dissociated neural cells, e.g., in the form of a cell suspension.
xe2x80x9cHibernation mediumxe2x80x9d as used herein, includes any medium which lacks a cryopreservative and is physiologically compatible for storage of a cell at above freezing temperatures, preferably about 4xc2x0 C.
xe2x80x9cCell suspensionxe2x80x9d as used herein includes cells in intact pieces of tissue that are contacted with a medium and cells which have been dissociated, e.g., by subjecting a piece of tissue to gentle trituration, which are in contact with a medium.
xe2x80x9cAdapted cell suspensionxe2x80x9d includes a cell suspension that has been stored above freezing, preferably at 40xc2x0 C., in hibernation medium for about 1 hour-5 days.
xe2x80x9cCryopreservation solutionxe2x80x9d includes a solution which contains a cryopreservative, i.e., a compound which protects cells against intracellular and/or cell membrane damage as the cells are frozen or thawed. A cryopreservative is identified by enhanced viability and/or functionality of cells in contact with the cryopreservative when compared with cells which are similarly frozen or thawed in the absence of the cryopreservative. Any cryopreservative can be used in conjunction with the instant methods and the term is meant to encompass both intracellular and extracellular cryopreservatives.
xe2x80x9cSuitable for transplantationxe2x80x9d refers to a cell or a population of cells which is stored, cryopreserved, and/or obtained using any of the instant methods and which are sufficiently viable and/or functional such that a neurological disorder or dysfunction is treated, e.g., one or more symptoms of a neurological disorder or dysfunction are ameliorated or reduced for a period of time following implantation of the cell or population of cells into a subject suffering from a neurological disorder or dysfunction.
In one aspect, the invention pertains to a method for storing a population of fetal porcine neural cells suitable for transplantation comprising: a) contacting a population of porcine neural cells with a hibernation medium to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4xc2x0 C. to thereby store a population of neural cells suitable for transplantation.
In another aspect the invention pertains to a method for cryopreserving a population of fetal porcine neural cells suitable for transplantation comprising: a) contacting a population of porcine neural cells with a cryopreservation solution to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to thereby cryopreserve a population of neural cells suitable for transplantation.
In yet another aspect, the invention pertains to a method of obtaining a population of fetal porcine neural cells suitable for transplantation comprising: a) contacting a population of porcine neural cells with a cryopreservation solution to thereby obtain a population of cells for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to obtain cryopreserved neural cells; c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation; and d) contacting the population of porcine neural cells suitable for transplantation with a hibernation medium and maintaining the cells at about 4xc2x0 C. prior to transplantation.
In one embodiment of the invention, the porcine neural cells are ventral mesencephalic cells. In a preferred embodiment, the ventral mesencephalic cells are porcine cells obtained between about days 25 and 28 of gestation. In another embodiment, the porcine cells are spinal cord cells. In another embodiment, the porcine neural cells are striatal cells. In a preferred embodiment, the striatal cells are obtained from a lateral ganglionic eminence of porcine striatum. In another embodiment, the porcine neural cells are cortical cells.
In another embodiment, the invention pertains to a population of porcine neural cells for transplantation prepared according to the instant methods.
In yet another embodiment, the invention pertains to a method for treating a neurological disorder or dysfunction comprising transplanting the population of porcine neural cells obtained using the instant methods into a subject.
In another aspect, the invention pertains to a method for storing a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium which medium is free of added protein to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4xc2x0 C. to thereby store a population of neural cells suitable for transplantation.
In another aspect, the invention pertains to a method for storing a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium which medium is free of a buffer to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4xc2x0 C. to thereby store a population of neural cells suitable for transplantation.
In another aspect, the invention pertains to a method for storing a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium which medium is free of added protein and free of a buffer to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4xc2x0 C. to thereby store a population of neural cells suitable for transplantation.
In another aspect, the invention pertains to a method for storing a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium which medium consists of glucose and sodium chloride to thereby produce a cell suspension; and b) maintaining the cell suspension at about 4xc2x0 C. to thereby store a population of neural cells suitable for transplantation.
In yet another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of added protein and which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to thereby cryopreserve a population of neural cells suitable for transplantation.
In another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of a buffer and which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells to about xe2x88x92196xc2x0 C. to thereby cryopreserve a population of neural cells suitable for transplantation.
In another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of added protein and free of a buffer and which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to thereby cryopreserve a population of neural cells suitable for transplantation.
In another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution consisting of glucose, sodium chloride, and a cryopreservative to thereby obtain a population of cells for cryopreservation; and b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to thereby cryopreserve a population of neural cells suitable for transplantation
In another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of added protein which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to obtain cryopreserved neural cells; and c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation.
In another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of a buffer and which comprises a cryopreservative to thereby obtain a population of cells for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to obtain cryopreserved neural cells; and c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation.
In yet another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution which is free of added protein and free of a buffer and which comprises a cryopreservative to thereby produce a population of neural cells suitable for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to obtain cryopreserved neural cells; and c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation.
In still another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a cryopreservation solution consisting of: glucose, sodium chloride, and a cryopreservative to thereby obtain a population of cells for cryopreservation; b) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C. to obtain cryopreserved neural cells; and c) increasing the temperature of the cryopreserved neural cells to thereby obtain a population of neural cells suitable for transplantation.
In certain embodiments of the invention, the neural cells are fetal human cells. In preferred embodiments, the neural cells are human neural stem or neural progenitor cells that have been induced to differentiate in vitro prior to storage using the instant methods.
In other embodiments of the invention, the neural cells are porcine cells. In still other embodiments, the porcine neural cells are ventral mesencephalic cells. In yet other embodiments, the porcine neural cells are porcine spinal cord cells. In still other embodiments, the porcine neural cells are striatal cells. In still other embodiments, the porcine neural cells are cortical cells. In still further embodiments, the porcine cells are porcine neural stem cells or neural progenitor cells. In certain embodiments, the porcine neural stem cells or progenitor cells have been induced to differentiate in vitro prior to storage using the instant methods.
In other embodiments of the invention, the invention pertains to a population of human or porcine neural cells for suitable for transplantation prepared according to the instant methods.
In still other embodiments of the invention, the invention pertains to a method for treating a neurological disorder or dysfunction comprising transplanting a population of human or porcine neural cells stored according to the claimed methods into a subject.
In another aspect, the invention pertains to a method for storing a population of porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium to thereby produce a cell suspension; b) maintaining the cell suspension for at least about 24 hours at about 4xc2x0 C. in hibernation medium to thereby store a population of neural cells suitable transplantation.
In another aspect, the invention pertains to a method for cryopreserving a population of human or porcine neural cells suitable for transplantation comprising: a) contacting a population of neural cells with a hibernation medium to thereby produce a cell suspension; b) maintaining the cell suspension for at least about 24 hours at about 4xc2x0 C. in hibernation medium to thereby produce an adapted cell suspension; c) contacting the adapted cell suspension with a cryopreservation solution to thereby obtain a population of cells for cryopreservation; and d) decreasing the temperature of the population of neural cells suitable for cryopreservation to about xe2x88x92196xc2x0 C. to thereby cryopreserve a population of neural cells suitable for transplantation.
In another aspect, the invention pertains to a method of obtaining a population of human or porcine neural cells for transplantation comprising: a) contacting a population of neural cells with a hibernation medium to thereby produce a cell suspension; b) maintaining the cell suspension for at least about 24 hours at about 4xc2x0 C. in hibernation medium to thereby produce an adapted cell suspension; c) contacting the adapted cell suspension with a cryopreservation solution to thereby obtain a population of cells for cryopreservation; d) decreasing the temperature of the population of neural cells for cryopreservation to about xe2x88x92196xc2x0 C.; and e) increasing the temperature of the neural cells to thereby obtain population of neural cells suitable for transplantation.
In certain embodiments the cell suspension is maintained at about 4xc2x0 C. for about 72 hours. In preferred embodiments the cell suspension is maintained at about 4xc2x0 C. for at least about 40-48 hours. In more preferred embodiments, the cell suspension is maintained at about 4xc2x0 C. for at least about 44 hours.
The invention is further described in the following subsections:
Neural cells useful in the methods of this invention can be fresh cells or may be obtained from in vitro culture.
Preferably, the cells of the invention are of mammalian origin, i.e., are obtained from mammalian subjects (e.g., humans, pigs, or cows). In one embodiment, the cells are bovine. Preferred cells for use in the instant methods are porcine. Other preferred cells are human.
Neural cells can be derived or obtained from a variety of tissues which are selected based, at least in part, on the intended use for the cells, e.g., the particular function to be assessed or clinical indication to be addressed. For example, if the cells are to be used to treat paralysis, it may be desirable to obtain them from the spinal cord of a donor subject. When the cells are intended for implantation into humans with Parkinson""s disease, they are preferably derived from a region of the brain giving rise to dopamine-producing cells.
Similarly, the neural cells useful in the methods of this invention may be obtained during various stages of development of the donor subject, including, fetal, juvenile, and adult cells. In general, the particular stage of development is selected based upon the intended use of the cells subsequent to storage and the species of animal from which the cells are derived.
In one embodiment, the cells for use in the present invention are fetal cells. Preferably, the cells are derived from the fetal central nervous system. In another embodiment, the fetal cells are spinal cord cells. In preferred embodiments the fetal cells are ventral mesencephalic cells. In still other preferred embodiments the fetal cells are striatal cells. In yet other preferred embodiments the striatal cells are obtained from a lateral ganglionic eminence of the striatum. In other embodiments, the fetal cells are cortical cells.
Preferably, cells for use in the instant methods are used after they have undergone their final maturation, but before they have sent out projections, e.g., axons. For example, in one embodiment, the fetal human cells are obtained from fetuses ranging in age from 7 to 18 weeks of gestation. In preferred embodiments, fetal human cells are obtained at between 7 and 11 weeks gestation. Fetal human cells for use in the claimed methods are obtained using methods known in the art and as required under the guidelines for use of human tissue (see e.g., DHEW publication OS 1975). In embodiments in which fetal porcine cells are used, preferably the cells are obtained between about days 20 and 115 of gestation, depending on the cell type to be isolated. For example, in certain embodiments, e.g., when the cells are porcine ventral mesencephalic cells, the cells are obtained between about days 25 and 28 of gestation. Preferably the porcine VM cells are used between about days 26 and 27 of gestation. More preferably, the porcine VM cells are used at about 27 days of gestation. In the case of fetal porcine striatal cells, preferably the cells are obtained from a fetus at between about days 30 and 50 of gestation. In more preferred embodiments, the porcine striatal cells are obtained from a fetus between about days 31 and 38 of gestation. In particularly preferred embodiments, the porcine striatal cells are obtained from a fetus between about days 34 and 36 of gestation. In the case of porcine cortical cells, the cells are preferably obtained from a fetus between about days 20 and 30 of gestation. In particularly preferred embodiments, the porcine cortical cells are obtained from a fetus between about days 24 and 28 of gestation.
In certain embodiments, the cells for use in the instant methods are neural stem cells which have been induced to differentiate. In other embodiments, the cells are neural progenitor cells which have been induced to differentiate. Tissue containing stem or progenitor cells can be obtained from mammalian fetuses, juveniles, or from an adult organ donor. In preferred embodiments, stem cells to be used in the instant methods are porcine cells. In other preferred embodiments, stem cells to be used in the instant methods are human cells. In certain embodiments, autologous stem cells from the donor may be obtained, differentiated and cryopreserved using the instant methods.
Neural stem or progenitor cells can be obtained from any area of the central nervous system, including the cerebral cortex, cerebellum, midbrain, brainstem, spinal cord, ventricular tissue, or from areas of the peripheral nervous system, including the carotid body and the adrenal medulla. Methods of obtaining neural progenitor or stem cells are known in the art (see e.g., U.S. Pat. No. 5,753,506; WO97/44442; WO96/04368; WO94/10292; WO94/02593; Gage et al. 1995 Ann. Rev. Neurosci. 18:159).
To expand a population of neural cells, (e.g., stem or progenitor cells) the cells can be grown in the presence of trophic factors, such as nerve growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, platelet-derived growth factor, thyrotropin releasing hormone, epidermal growth factor, amphiregulin, transforming growth factor, transforming growth factor xcex2, insulin-like growth factor, or other growth factors using methods known in the art (see, e.g., U.S. Pat. No. 5,753,506, U.S. Pat. No. 5,612,211, U.S. Pat. No. 5,512,661, WO93/01275; Mehier and Kessler. 1995 Crit. Rev. Neurobiol. 9:419).
Neuronal or glial cells can be differentiated from an expanded stem or progenitor cell population by treating the cells by any method known in the art which promotes differentiation of the cells, for example, phorbol esters or various growth factors (See, e.g., U.S. Pat. Nos. 5,750,376; 5,753,506; WO 96/15226; WO 94/02593; WO 96/15224). Alternatively, a surface such as poly-L-lysine can be used to induce differentiation (WO 93/01275). Such differentiated cells can be stored using the instant methods.
In certain embodiments, pieces of tissue are left intact, or stored as fragments, e.g., are not dissociated into individual cells prior to used in the instant methods. In preferred embodiments, the cells to be used in the claimed methods are dissociated, e.g., into individual cells to make a suspension prior to their use in the instant methods. For example, tissue can be dissociated using gentle trituration. Methods of dissociating tissue are known in the art and include the use of flame-polished pasteur pipets of diminishing orifice diameters or a succession of Angio-catheters of diminishing orifice diameters.
Numerous types of media can be used as hibernation media in conjunction with the instant methods. In preferred embodiments, hibernation media is free of added Ca++. In certain embodiments, medium for hibernating cells is free of added protein and/or free of a buffer. A preferred hibernation medium includes or consists of glucose in a saline solution, e.g., between about 0.2%-0.9% glucose in saline. In preferred embodiments, the hibernation medium includes or consists of about 0.35-0.9% glucose and 0.9% NaCl. In other preferred embodiments, the medium includes or consists of about 0.6% glucose and 0.9% NaCl. In certain embodiments, more complex media can be used, e.g., Hank""s balanced salt solution, Dulbecco""s minimal essential medium (see e.g., Nikkah et al. 1995 Brain Research 687:22), or Eagle""s modified minimal essential medium. Another suitable hibernation medium has been described by Kawamoto and Barrett (1986 Brain Research 384:84). In certain embodiments it may be desirable to supplement the chosen hibernation medium with additives, for example, added protein (e.g., mammalian serum protein or whole serum (preferably heat inactivated)) buffers (e.g., phosphate buffers, HEPES, or the like) antioxidants, growth factors, KCl (e.g., at about 30 mM), lactate (e.g., at about 20 mM), pyruvate, MgCl2 (e.g., at about 2-3 mM), sorbitol (e.g., at about 300 mM) or other additives as are well known in the art (e.g., as taught by Kawamoto and Barrett supra).
In certain embodiments, the cells of the invention are hibernated at about 0-37xc2x0 C., preferably about 40xc2x0 C. In certain embodiments, cells are maintained at about 4xc2x0 C. in hibernation medium prior to freezing or use. In other embodiments the cells of the invention are maintained at about 4xc2x0 C. in hibernation medium post freezing. In still other embodiments, the cells of the invention are maintained at about 4xc2x0 C. in hibernation medium without freezing. In certain embodiments, the cells of the invention are maintained in hibernation medium at about 4xc2x0 C. for at least about 1 hour to 5 days prior to freezing, post freezing or prior to use in transplantation. In other embodiments, the cells of the invention are maintained in hibernation medium at about 4xc2x0 C. for at least about 12-72 hours prior to freezing, post freezing or prior to use in transplantation. In certain embodiments the cells are maintained at 4xc2x0 C. in hibernation medium for at least about 24 hours prior to freezing, post freezing or prior to use in transplantation. In a more preferred embodiment, the cells are maintained in hibernation medium from at least about 40-48 hours at about 4xc2x0 C. prior to freezing, post freezing or prior to use. In a particularly preferred embodiment, the cells are maintained in hibernation medium for at least about 44 hours at about 4xc2x0 C. prior to freezing, post freezing or prior to use.
Any cryopreservative known in the art can be used in a cryopreservative solution of the instant invention. In preferred embodiments, cryopreservation solutions of the present invention include intracellular cryopreservatives e.g., dimethylsulfoxide (DMSO), various diols and triols (e.g., ethylene glycol, propylene glycol, butanediol and triol and glycerol), as well as various amides (e.g., formamide and acetamide). However, extracellular cryopreservatives e.g., phosphomono and phosphodiester catabolites of phosphoglycerides (EP 0 354 474), polyvinylpyrrolidone (Fang and Zhong 1992 Cryobiology 29:267), or methylcellulose (e.g., at 0.1%, see e.g., Sautter et al. 1996 J. of Neuroscience Methods. 64:173) can also be used alone or in conjunction with an intracellular cryopreservative.
In preferred embodiments, DMSO is used as the cryopreservative. DMSO can be used at a wide range of concentrations (see e.g., Silani et al. 1988 Brain Research 473:169). In preferred embodiments, DMSO is used at a final concentration of about 4% to about 10%. In more preferred embodiments the concentration of DMSO ranges from about 7% to about 10%. In particularly preferred embodiments the concentration of DMSO is about 7%.
In certain embodiments, the cryopreservative is added to the cells in a stepwise manner in order to gradually increase the concentration of the cryopreservative until the desired final concentration of cryopreservative is achieved. In preferred embodiments, the cells are contacted with a cryopreservation solution containing the cryopreservative at the desired final concentration or the cryopreservative is added directly to the base medium without a gradual increase in concentration.
The cryopreservation solution includes the cryopreservative in an appropriate base medium. Any type of media can be used for this purpose. For example, any of the hibernation media listed above can be used as the base medium for a cryopreservation solution. In preferred embodiments, the base medium to which the cryopreservative is added is free of added Ca++. In certain embodiments the medium to which the cryopreservative is added is free of added protein and/or free of a buffer. In other embodiments, the base medium to which the cryopreservative is added includes or consists of about 0.2-0.9% glucose and about 0.9% NaCl. In preferred embodiments, the base medium to which the cryopreservative is added includes or consists of about 0.35-0.9% glucose and about 0.9% NaCl. In another preferred embodiment, the base medium to which the cryopreservative is added includes or consists of about 0.6% glucose and about 0.9% NaCl. Alternative media can also be used, see e.g., that described by Gage et al. (1985 Neuroscience Letters 60:133).
In certain embodiments the cryopreservation solution can also contain added protein, for example, serum, e.g., fetal calf serum or human serum, or a serum protein, e.g., albumin. In other embodiments, the cryopreservative can also contain other additives, such as those described above for inclusion in hibernation media, for example, antioxidants, growth factors, KCl (e.g., at about 30 mM), lactate (e.g., at about 20 mM), pyruvate, MgCl2 (e.g., at about 2-3 mM), sorbitol (e.g., to an osmolarity of about 300 mM) or other additives as are well known in the art (e.g., as taught by Kawamoto and Barrett supra).
Once the cells are suspended in cryopreservation solution, the temperature of the cells is reduced in a controlled manner. In cooling the cells to below freezing, preferably the reduction in temperature occurs slowly to allow the cells to establish an equilibrium between the intracellular and extracellular concentration of cryopreservative such that intracellular ice crystal formation is inhibited. On the other hand, the rate of cooling is preferably fast enough to protect the cells from excess water loss and the toxic effects of cryopreservatives. Controlled freezing may be accomplished with the aid of commercially available electronically controlled freezer equipment, e.g., a Cryomed or Planer Biomed controlled rate freezer. In an exemplary freezing program a cell sample and the freezing chamber are brought to about 1xc2x0 C. to 9xc2x0 C., preferably about 4xc2x0 C. The cells are then cooled at a rate of about 1xc2x0 C./min to 7xc2x0 C./min, preferably about 2xc2x0 C./min. until the cells reach about xe2x88x926xc2x0 C. to +6xc2x0 C., preferably about xe2x88x921xc2x0 C. The chamber is brought to about xe2x88x926xc2x0 C. to +6xc2x0 C., preferably about xe2x88x921xc2x0 C., and the sample is held at this temperature for about 9-19 minutes, preferably about 14 minutes. The sample is cooled at a rate of about 1xc2x0 C./minute to 6xc2x0 C./minute, preferably about 1.2xc2x0/minute until the sample reaches about xe2x88x9216xc2x0 C. to about xe2x88x926xc2x0 C., preferably about xe2x88x9211xc2x0 C. The freezing chamber is subsequently cooled at a rate of about 83 to about 92xc2x0 C./minute, preferably about 88xc2x0/minute until the sample reaches about xe2x88x9222xc2x0 C. to xe2x88x9232xc2x0 C., preferably about xe2x88x9227xc2x0 C. The chamber is warmed to about xe2x88x9238 to about xe2x88x9228xc2x0 C., preferably about xe2x88x9233xc2x0 C. and the sample is held for about 5-15 minutes, preferably about 10 minutes. The cells are then cooled at a rate of about 1-5xc2x0/minute, preferably about 1xc2x0/minute until the sample reaches about xe2x88x9240 to about xe2x88x9250xc2x0 C., preferably about xe2x88x9245xc2x0 C. Next, the cells are cooled at about 1-7xc2x0/minute, preferably about 2xc2x0/minute until the cells reach about xe2x88x9255 to about xe2x88x9265xc2x0 C., preferably about xe2x88x9260xc2x0 C. The sample is then cooled at about 1xc2x0 to about 10xc2x0/minute, preferably about 5xc2x0/minute until it reaches xe2x88x9290xc2x0 C.
The cells can then be cryopreserved at a temperature of between xe2x88x9220xc2x0 C. and about xe2x88x92250xc2x0 C. Preferably, the cells are stored below xe2x88x9290xc2x0 C. to minimize the risk of ice recrystalization. In particularly preferred embodiments, the cells are cryopreserved in liquid nitrogen at about xe2x88x92196xc2x0 C.
After cryopreservation, the cells are preferably thawed rapidly, e.g., by quick immersion in liquid at 37xc2x0 C. Once the cells are thawed, dilution of the cryopreservative is accomplished by gradual addition of a dilution media. Preferably, the cryopreservative is gradually diluted by a slow multi-step addition of media. For example, cells can be diluted slowly by adding a dilution medium (e.g., a 1:1 dilution) and allowed to sit for 5 minutes at room temperature. The 1:1 dilution can be repeated twice more by slowly adding dilution medium, waiting 5 minutes, and then adding more dilution medium. In other embodiments, a one-step dilution procedure can be used.
Any media can be used for diluting the cryopreservation solution which is in contact with the thawed cells. For example, any of the media listed above for use in hibernating cells can be used for diluting the cryopreservation solution. Other media are also appropriate, for example, Hank""s balanced salt solution (preferably without Ca++) containing glucose (about 0.6%) can be used. Additives, e.g., as listed above for inclusion in hibernation or freezing media can also be used in media for dilution. Exemplary additives include, for example, buffers (e.g., phosphate buffers, HEPES, or the like) antioxidants, growth factors, KCl (e.g., at about 30 mM), lactate (e.g., at about 20 mM), pyruvate, MgCl2 (e.g., at about 2-3 mM), sorbitol (e.g., to an osmolarity of about 300 mM) or others additives as are well known in the art (e.g., as taught by Kawamoto and Barrett supra). Another suitable additive includes DNase (e.g., commercially available from Genentech, Incorporated as PULMOZYMEOR(copyright)). The medium which is used for diluting the cryopreservation solution can, optionally, contain added protein, e.g., added protein (e.g., mammalian serum (preferably heat inactivated) or a serum protein such as albumin (e.g., commercially available from Alpha Therapeutic Corporation)). In other embodiments, the medium contains no added protein and/or no added buffer.
When cells have been frozen as pieces of tissue, the thawed tissue can be dissociated, if desired, into individual cells, using methods known in the art and described supra.
After dilution of the cryopreservative, the cells can then be allowed to settle or a pellet of cells can be formed under centrifugal force in order to remove as much of the cryopreservation solution from the cells as possible. The cells can then be washed in medium which does not contain a cryopreservative. It is preferable for the cells to remain at room temperature after the addition of the wash media and prior to letting the cells settle or form a pellet under centrifugal force. In preferred embodiments, the cells remain at room temperature for at least 15 minutes prior to the second centrifugation. Any medium known in the art can be used to wash the cells, for example, any of the hibernation or dilution media set forth above can be used.
For use in transplantation, cells should be suspended in a final medium which is suitable for administration to a subject. In certain embodiments, the cells are resuspended in a solution including or consisting of glucose (e.g., about 0.2-0.9%) and sodium chloride (e.g., about 0.9%). In preferred embodiments, the cells are resuspended in a solution including or consisting of glucose (e.g., about 0.3-0.6%) and sodium chloride (e.g., about 0.9%). In particularly preferred embodiments, the cells are resuspended in a final solution including or consisting of about 0.35% glucose and about 0.9% saline
In addition, the thawed cells may be maintained in hibernation medium as described above at between 0 and 37xc2x0 C., preferably about 4xc2x0 C. for up to 3-5 days prior to use in transplantation without a statistically significant loss in viability.
Methods of Determining Viability and/or Functionality of Recovered Cells
After storage, it may be desirable to assay the viability and/or functionality of the cells prior to transplantation to confirm their suitability for use, e.g., in transplantation. This can be accomplished using a variety of methods known in the art. For example, the cells can be stained using vital stains, such as, e.g., trypan blue or ethidium bromide or acridine orange. In certain embodiments, a population of cells suitable for transplantation is at least between about 75-100% viable. In preferred embodiments, a population of cells suitable for transplantation is at least about 80% viable. In particularly preferred embodiments, such a population of cells is at least about 85% viable.
In other embodiments, the morphometric characteristics of the cells can be determined as a measure of the suitability of cells for use in transplantation (see e.g., Petite and Calvet. 1997 Brain Research 769:1). In preferred embodiments, the morphology of cells which have been stored using the instant methods and are suitable for transplantation does not differ (e.g., statistically significant) from that of fresh cells. The morphology of the cells and their ability to integrate into the host nervous system can also be tested post-transplantation (Nikkhah et al. 1995 Brain Research 687:22). In preferred embodiments, the in vivo morphology of cells which have been stored using the instant methods and are suitable for transplantation does not differ (e.g., statistically significant) from that of fresh cells. Graft volume of transplanted cells can also be measured (Sauer and Brundin. 1991 Restorative Neurology and Neuroscience 2:123). In preferred embodiments, the graft volume of cells which have been stored using the instant methods and are suitable for transplantation does not differ (e.g., statistically significant) from that of fresh cells.
Cells which have been stored can also be assayed for the presence of neural cell markers to determine if they are suitable for use in transplantation. For example, methods and reagents useful in detecting the expression of glial fibrillary acidic protein, gamma amino butyric acid (GABA), neuron-specific enolase, tyrosine hydroxylase (TH), norepinephrine, serotonin, 3,4,-dihydroxyphenylacetic acid (DOPAC), homovanillic acid, 5-hydroxyindole acetic acid, acetyl cholinesterase, or other markers are available (see, e.g., Petite and Calvet 1995 Brain Research 669:263; Collier et al. 1987 Brain Research 436:363; Petite and Calvet 1997 747:279). In preferred embodiments, cells suitable for transplantation display an immunoreactivity pattern, e.g., TH activity, which is not lower (e.g., statistically significant) than that demonstrated in a fresh population of cells.
Additionally, or alternatively, the cells can be tested for their functionality. For example, the ventral mesencephalon cells could be transplanted into 6-OHDA lesioned rats (Kamo et al. 1986 Brain Research 397:372). The ability of the cells to reduce pregraft ipsilateral amphetamine-induced motor asymmetry can be tested. In preferred embodiments, a population of cells suitable for transplantation compensates (e.g., statistically significant) for a neural defect in such an in vivo animal model. For example, a population of cells obtained by the instant methods compensates for a 6-OHDA lesion as well as or better than (e.g., statistically significant) a fresh population of cells.
Cells which have been cryopreserved using the instant methods can be used to treat a variety of neurodegenerative diseases or dysfunctions. For example, Parkinson""s disease, Huntington""s disease, Lou Gehrig""s disease or amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer""s disease, have all been linked to the degeneration of neurons in specific locations in the brain or spinal cord. In addition, damage to the nervous system, caused by, for example, stroke, epilepsy, cerebral palsy, spinal cord injury, or chronic pain all result in neuronal loss and can be treated to restore neuronal physiology using cells obtained by the instant methods.
This invention is illustrated further by the following examples which should not be construed as further limiting the subject invention. The contents of all references, published patent applications and patents cited throughout this application are incorporated herein by reference.